1. INTRODUCTION:

Salbutamol sulphate (SAL), chemically known as bis [(1RS)-2-[(1, 1-dimethylethyl) amino]-1-[4-hydroxy-3-(hydroxymethyl) phenyl] ethanol] sulphate, is beta-adrenocepter agonist used for the relief of Broncho-spasm in conditions such as asthma and chronic obstructive pulmonary disease and it is official in Indian pharmacopoeia^1,2Chemically Prednisolone(PRE) is a glucocorticoid and its IUPAC name is (8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl7,8,9,11,12,14,15,16-octahydro-6H-cyclopenta[a]phenanthren-3-one.

Prednisolone is used as anti-inflammatory or immunosuppressiveagent³ and it is official in India pharmacopoeia. Both the drugs alone or in combination with other drugs are reported to be estimated but do not involve simultaneous determination of SAL and PRE.

Detailed survey of literature for SAL revealed several methods based on different techniques such as UV Spectrophotometry^4-8, RP-HPLC⁹, and TLC¹⁰ for its determination from pharmaceuticals. Similarly survey of literature for PRE alone or in combination with other drugs is reported to be estimated by UV Spectrophotometry¹¹, RP-HPLC¹², matrix solid phase dispersion liquid chromatography ^[13], and LC-MS ^[14] for its determination from pharmaceuticals. But no methods have been reported for simultaneous determination of SAL and PRE. Hence in the present work a successful attempt has been made to estimate both these drugs simultaneously by UV spectrophotometric method (Area under curve method^[^{15, 16}^] and HPTLC method. To establish stability indicating nature ^[17] of the TLC method, forced degradation of drug substances was performed under stress conditions (acid and base hydrolysisoxidation). The proposed methods were optimized and validated as per ICH guidelines.

2. EXPERIMENTAL:

2.1 Chemicals:

Gift samples of Salbutamol sulphate and Prednisolone were provided by Macleods Pharmaceutical Pvt.Ltd Mumbai (Maharashtra) and Lupin laboratories Ltd. Pune (Maharashtra) respectively. All chemicals and reagents used were of AR grade.

2.2 Instrumentation and Chromatographic Conditions:

The UV method was performed on a Double-beam Shimadzu UV- Visible spectrophotometer, 1700, with spectral bandwidth of 2 nm, wavelength accuracy ± 0.5 nm and a pair of 1-cm matched quartz cells was used to measure absorbance of solution. Working wavelength for UV method was 232-222nm and 249-239 nm (Figure.1.)

Figure No.1: Spectrum SAL and PRE from 200 to 400 nm.

he standard solution ranging from 1000-6000ng/band was applied on precoated silica gel 60 F₂₅₄TLC plate in the form of bands with 100 µl sample syringe using automatic sample applicator LINOMAT V. It was developed in a twin trough glass chamber which was already saturated for 30 min. with the mobile phase. The mobile phase consisted of Ethyl acetate: Chloroform: methanol (6.0: 4.0: 1.0 v/v/v). After development, plate was immediately dried and was observed under UV chamber. The well resolved bands of drug were scanned at 230 nm with Camag TLC scanner III densitometer controlled by WINCAT’s software version 4.

2.3 Preparation of standard solutions:

2.3.1. UV method:

Standard stock solution containing SAL and PRE were prepared by dissolving quantity of Salbutamol sulphate equivalent to SAL base 2.5mg and 2.5mg of PRE separately in 20ml of methanol in separate 25ml volumetric flask and final volume of both solutions were made up to 25ml with methanol to get stock solution containing each of 100µg/ml of SAL and PRE. From these solutions, concentrations of 6-20 µg /ml were made in 10.0ml volumetric flasks.

2.3.2. HPTLC method:

Standard stock solution of SAL and PRE were prepared by dissolving quantity of Salbutamol sulphate equivalent to SAL base 5.0mg and 5.0mg of PRE separately in methanol in separate 10.0ml volumetric flask and final volume of both solutions were made up to mark with methanol to get a stock solution of 500µg/ml.

2.4. Preparation of the sample solutions:

2.4.1. UV method:

Twenty tablets were weighed and average weight was calculated. The tablets were crushed to obtain fine powder. Tablet powder equivalent to 2.0 mg of PRE and 1.6mg of SAL was transferred to 10.0 ml volumetric flask; 5 ml methanol was added and sonicated for 10 min. The volume was then made up to the mark with methanol. The resulting solution was filtered through Whatmann filter paper and filtrate was appropriately diluted to get approximate concentration of 8μg/ml of PRE and 6.4μg/ml of SAL.

2.4.2. HPTLC method:

Twenty tablets were weighed and average weight was calculated. The tablets were crushed to obtain fine powder. Tablet powder equivalent to 4.0mg SAL and 5.0mg of PRE was taken and dissolved in mobile phase and sonicated for 20min. and then volume was made up to the mark with mobile phase. The resulting solution was mixed and filtered through Whatmann filter paper No. 41. From the filtratesample solution were applied six times on TLC plate to give spot concentration 1600ng/band of SAL and 2000 ng/band of PRE. The plate was developed in the previously described chromatographic conditions. The peak areas of the spots were measured at 230 nm and concentrations in the samples were determined using multilevel calibration.

2.5. Method Validation:

The method was validated in compliance with ICH guidelines¹⁸.

2.6. Procedure for forced degradation study:

For forced degradation studies, samples were prepared by addition of 1 ml of 0.1N hydrochloric acid, 0.1N sodium hydroxide and 3% hydrogen peroxide to sample solutions in separate volumetric flasks and were diluted with methanol to get the final concentration of 1600ng/band of SAL and 2000ng/band of for acid, alkali, hydrogen peroxide induced degradation respectively. These solutions were allowed to stand for 24 hrs and drug solution containing 1600ng/band of SAL and 2000ng/band of PRE was exposed to sunlight for 2 hrs for degradation.

Figure No.2: Densitogram of SAL (1600 ng/band, Rf = 0.17) andPRE (2000 ng/band, Rf = 0.52)

Table No.1 Result of analysis of formulation

Method	Drug	Label Claim ( mg/Tablet)	% of Label Claim Estimated*	(±)Standard Deviation	% R.S.D.
UV Spectrophotometry	SAL	4.0	101.18	0.8564	0.8463
UV Spectrophotometry	PRE	5.0	101.82	0.2049	0.2012
HPTLC	SAL	4.0	101.26	0.827	0.8167
HPTLC	PRE	5.0	99.53	0.5818	0.5845

* Mean of six estimation

Table No.2.Result of recovery studies

Level of recovery	Drug	UV method			HPTLC method
Level of recovery	Drug	% Recovery*	(±)Standard deviation*	%RSD*	% Recovery*	(±)Standard deviation*	%RSD*
80	SAL	101.16	0.5973	0.5904	101.32	0.6701	0.6613
80	PRE	101.54	0.6200	0.6105	100.33	0.8890	0.8860
100	SAL	101.31	0.6243	0.6223	98.82	0.1415	0.1431
100	PRE	101.98	0.7170	0.7006	100.04	0.3789	0.3797
120	SAL	100.89	0.2974	0.2947	100.89	0.7916	0.7846
120	PRE	101.47	0.0838	0.0826	101.43	0.2444	0.2409

* Mean of three estimation.

3. RESULTS AND DISCUSSION:

3.1. Optimization of Procedures:

Different proportions of ethyl acetate, chloroform and methanol were tried while selection of mobile phase. Ultimately ethyl acetate: chloroform: methanol (6.0:4.0:1.0 %v/v/v) was finalized as mobile phase. The spots developed were dense, compact and typical peak of SAL and PRE obtained was shown in Figure No.2. Peak was symmetrical in nature and no tailing was observed when plates were scanned at 230 nm.

3.2. Linearity:

For UV spectroscopy the analytical concentration range over which the drugs obeyed Beer Lambert’s law was found to be 6-20µg/ml for SAL and PRE and the coefficient of regression for both the drugs was found to be nearer to 1. For HPTLC Calibration curves were constructed in the concentration range of (1000-6000ng/band for SAL and 2000-6000ng/band for PRE). Beer’s law is obeyed over this concentration range, and the coefficient of regression for both the drugs was 0.9966 and 0.9952 respectively.

3.3. Analysis of Formulation:

In UV spectroscopy two simultaneous equations (in two variables C_SAL and C_PRE) were formed using absorptivity coefficient values and these are as follows.

A_{1(at λ 232nm-222nm)} = (0.3338) C_X + (0.3011) C_Y …….. (I)

A_{2 (AUC at
249-239nm)} = (0.0029) C_X+ (0.4514) C_Y………..(II)

By solving above simultaneous equation the concentration C_X(Salbutamol)and C_Y(Prednisolone) was calculated. Table.1. shows the experimental values obtained for the determination of SAL and PRE in samples.

In HPTLC the spots at Rf 0.17 and 0.52 for SAL and PRE were observed respectivelyin the densitogram of the drug samples extracted from tablets. There was no interference from the excipients commonly present in the tablets. The SAL and PRE content was found to be close to 100% and the results are summarized in Table No 1. The low %RSD value indicated the suitability of this method for routine analysis.

3.4. Precision:

Precision was evaluated by carrying out six independent sample preparation of a single lot of formulation. Percentage relative standard deviation (%RSD) was found to be less than 2% for inter day and intraday variations for the both method.

3.5. Recovery Studies:

To check the degree of accuracy of the method, recovery studies were performed in triplicate by standard addition method at 80%, 100% and 120%. Known amounts of standard SAL and PRE were added to pre-analyzed samples and were subjected to the proposed method. Results of recovery studies for the bothmethods are shown in Table No 2.

3.6. Robustness:

The robustness of the HPTLC method was determined by variations in methanol composition (± 2%), chamber saturation period (± 10%), development distance (± 10%), time from application to development (0, 10, 20, 30 min), time from development to scanning (0, 10, 20, 30 min). One factor at a time was changed at a concentration level of 1600 ng/band and 2000 ng/band for SAL and PRE respectively, to study the effect on the Rf and peak area of the drugs. The method was found to be unaffected by small changes with % RSD for all the parameters less than 2% indicating that method is robust.

3.7. Stability-Indicating Property:¹⁷

The stability study of SAL and PRE under different stress conditions was carried out.SAL and PRE was underwent acid hydrolysis, but the rate of hydrolysis was slower as compared to that in alkali. It was depicted in Fig.3.

Figure No 3: Chromatogram of Acid treated sample.

The degradation reaction was more intense and quicker in alkaline condition. And decrease in peak area of both the drug was observed and it was shown in Fig.4.

Figure No 4: Chromatogram of Alkali treated sample.

Oxidative degradation as depicted in Fig.5, more degradation of SAL as compared to PRE was seen and decrease in the peak area of both the drugs was observed.

Figure No 5: Chromatogram of Hydrogen peroxide treated sample

A cluster of peaks was observed along with the peak of SAL and PRE in acid and base hydrolysis, which represents probable degradation of pure drugs and needs further investigation. When drug substance exposed to sun light complete degradation of PRE was seen as depicted in Fig.6.

Figure No 6: Chromatogram of sample exposed to sunlight.

The amount of drug recovered after degradation studies and the Rf of degradation products are given in Table No 3.

Table No. 3. Results of forced degradation studies

Stress condition	Time (hours)	% Assay of active substance		R_f values of degradation products
Stress condition	Time (hours)	SAL	PRE	R_f values of degradation products
Acid hydrolysis (0.1 N HCl)	24	88.31	82.19	0.19
Base hydrolysis (0.1N NaOH)	24	76.29	77.99	0.21,0.41
Oxidation (3% H₂O₂)	24	90.01	96.81	-
Exposed to sunlight	1.0	82.82	8.55	0.27,0.41,0.59

4. ACKNOWLEDGEMENT:

The authors are thankful to Dr. Avinash D. Deshpande, Director of Pharmacy, and Padm.Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune for providing necessary facilities and to Macleods Pharmaceutical Pvt.Ltd Mumbai (Maharashtra) and Lupin laboratories Ltd. Pune (Maharashtra) for providing gift sample of pure drug.

5. CONCLUSION:

The proposed UV spectrophotometric and TLC-densitometric method was validated as per ICH guidelines. The standard deviation, %RSD and standard error calculated for the method are low, indicating high degree of precision of the method. The results of the recovery studies performed shows high degree of accuracy of the proposed method. The results of the stress studies indicated the specificity of the method. The method gives well-resolved peaks of SAL and PRE even after exposure to different stress conditions. The method can be used to determine the purity of the drugs available from various sources by detecting the related impurities. As the method is stability indicating one it may be extended to study the degradation kinetics of SAL and PRE.

Hence, it can be concluded that the developed UV spectrophotometric and TLC-densitometric method is accurate, precise, and selective and can be employed successfully for the estimation of SAL and PRE in bulk and in pharmaceutical formulation.

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Received on 28.02.2011 Modified on 08.03.2011

Asian J. Research Chem. 4(5): May, 2011; Page 786-790